The present invention relates to an immunoassay method for measuring a specific component in a sample.
Conventionally, various immunoassay methods using antibodies have been proposed as methods for measuring a specific component (subject substance) in a sample easily without involving dilution or agitation of a sample liquid.
Immunoturbidimetry is known as one of the immunoassay methods. An assay kit used for immunoturbidimetry (Microalbumin-HA TestWako of Wako Pure Chemicals Industries, Ltd) is commercially available. This assay kit is essentially composed of a buffer (50 mM Good's buffer, pH 7.4) and an antibody solution. The antibody solution includes a rabbit-derived anti-human albumin polyclonal antibody (1.5 mg Ab/ml) and a mouse-derived anti-human albumin monoclonal antibody (4.0 mg Ab/ml). In measurement, a sample is allowed to act with the antibody solution in the buffer. If albumin is included in the sample, the albumin specifically reacts with the anti-human albumin antibodies as an antigen-antibody reaction, turning the solution turbid. The degree of being turbid (turbidity) is proportional to the concentration of albumin in the sample. By measuring the turbidity, therefore, the albumin amount in the sample can be determined.
The reason why the assay kit described above uses both the polyclonal antibody and the monoclonal antibody is considered to combine the feature of the former of “enhancing the turbidity” and the feature of the latter of “causing less easily a prozone phenomenon”. That is, the combination of the polyclonal antibody and the monoclonal antibody for simultaneous use can broaden the measurable range while preventing occurrence of the prozone phenomenon in an antigen excess region, which is a shortcoming of the immunoturbidimetry (see K. Hatsuta et al., The 28th Japan Society of Clinical Chemistry Meeting Proceedings, 63, 1988, p. 125).
The immunoturbidimetry described above theoretically permits measurement of various substances, by selecting an antibody binding to a subject substance to be measured for each subject substance. For example, if an anti-human albumin antibody is selected as the antibody, a sensor for measuring the concentration of albumin in a sample liquid can be produced.
Albumin is a protein occupying about 50% of the protein in plasma that is the remainder of blood obtained after removing solid components such as blood cells. Albumin is known to have functions such as maintenance of the osmotic pressure of blood, carrying of ions, fat acids, part of vitamins, pigments and drugs, and supply of amino acids to peripheral tissues. However, albumin is also known to exude to urine in an event of a renal function disorder caused by diabetic nephropathy, one of diabetic complications, and the like.
By measuring the albumin amount in urine, therefore, screening of morbidity, check of the effect of a therapy, determination of prognosis and the like are allowed. For this reason, in recent years, measurement of the albumin amount in urine has become widespread as an important routine test for early detection of diabetic nephropathy and diagnosis of prognosis of diabetes. Measurement of the albumin amount in urine can be executed by an immunological technique using an antibody specifically binding to albumin.
As an antibody against albumin, an anti-human albumin serum as a polyclonal antibody has been conventionally used. In most of conventional albumin measurement methods, albumin is measured by determining whether or not a precipitation line exists, or the turbidity has increased, due to agglutination of the albumin with an antiserum. However, an unpurified antiserum, which often includes various antibodies against various antigens in a serum, may possibly bind to an antigen other than albumin. In the measurement, therefore, to prevent the antiserum from binding to an antigen other than albumin mistakenly, the antiserum is purified, to thereby block occurrence of any reaction derived from an antigen other than albumin in a human standard serum. For purification of an antiserum, a technique such as affinity chromatography is normally used in which an antibody likely to bind to a protein in a human standard serum is removed from the unpurified antiserum.
In the immunoturbidimetry described above, a polyclonal antibody (that is, an antiserum) is used. A polyclonal antibody is generally obtained by purifying a serum collected from blood of an immunized animal. Therefore, although the production method is easy, the properties of the resultant antibody may be highly possibly influenced by the family, environment, physical condition and the like of the immunized animal. For example, in the case of using an anti-human albumin serum, the kind of an anti-human albumin antibody included in an anti-human albumin serum produced may possibly be different (for example, the epitope may be different) from others, and as a result, the reactivity with albumin may vary every time an anti-human albumin serum is produced.
If the reactivity of a polygonal antibody varies every time a new polyclonal antibody is produced, it is difficult to obtain homogeneous antibodies continuously. In this situation, measurement of a subject substance by the immunoturbidimetry may fail to provide repeatable results.